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Formulation of Entomopathogenic Nematodes for Crop Pest Control – a Review

Heriberto Cruz-Martínez 1,2 *, Jaime ruiz-Vega3, Pastor t. MatadaMas-Ortíz 2, Carlos i. COrtés-Martínez 3 and Jesusita rOsas-diaz 3

1Programa de doctorado en nanociencias y nanotecnología, CinVestaV, san Pedro zacatenco, gustavo a. Madero, d.F., México; 2departamento de ingeniería and

3departamento de Protección Vegetal, instituto Politécnico nacional, Ciidir-Oaxaca, noche Buena, santa Cruz Xoxocotlán, Oaxaca, México

*Corresponding author: hcruzm@cinvestav.mx

Abstract

Cruz-Martínez H., Ruiz-Vega J., Matadamas-Ortíz P.T., Cortés-Martínez C.I., Rosas-Diaz J. (2017): Formulation of entomopathogenic nematodes for crop pest control – a review. Plant Protect. Sci., 53: 15–24.

The different materials and methods used to develop biopesticides with entomopathogenic nematodes (EPNs) still limit the quality of the final product, reducing field efficacy and complicating application strategies. Therefore, the objective of this review is to identify priority areas to improve the EPN formulation process based on the scientific and technological research developed so far. The results show great progress in the EPN survival time, from 7 days to 180 days, through two types of formulations: water dispersible granules and calcium alginate capsules. Also, EPNs formulated and applied as insect cadavers showed higher efficacy for the pest control than the EPNs applied in an aqueous solution. We consider that the priority areas of research are: automated massive formulation and explora-tion of new additives with better properties that may lead to increase the infectivity in the field. It is concluded that the study of these aspects requires a systemic approach with greater involvement of the invertebrate physiology and systems modelling disciplines.

Keywords: steinernema spp.; Heterorhabditis; insect cadavers; pellets, survival time

The excessive use of chemical pesticides in ag-riculture causes serious damage to soil, air, water, flora, fauna and human beings (Georgis 1992; Vos et al. 2000). Therefore, it is necessary to develop environmentally friendly alternatives to control soil pests, such as entomopathogenic nematodes (EPNs). The EPNs of the families Heterorhabditidae and Steinernematidae are obligate parasites of insects and have demonstrated to pose a high potential for the agricultural pest control (Ehlers 1996; Georgis et al. 2006; Lacey & Shapiro-Ilan 2008; San-Blas 2013; Tofangsazi et al. 2014; van Zyl & Malan 2014; Půža 2015). Also, they have been exempted from registration in the USA, are compatible with many chemical pesticides and amenable to genetic

selection (Kaya & Gaugler 1993). These EPNs pos-sess a symbiotic association with pathogenic bacteria of the genera Xenorhabdus and Photorhabdus, which are associated with steinernema and Heterorhabditis, respectively (Poinar 1990). The infective juveniles (IJs) are the only free-living stage and serve three main functions: dispersal, host finding, and survival under environmental detrimental conditions (Gre-wal et al. 2006). When the IJs enter the body cavity of a host-insect, the symbiotic bacteria are released, multiply and the host death occurs within two days (Poinar & Grewal 2012). The IJs complete their development and reproduce for two or three gen-erations inside their host. When food is depleted, a new generation of IJs exits from the host cadaver

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to search new hosts (Grewal & Georgis 1999; Shapiro-Ilan et al. 2012).

Currently, the factors that affect the application of EPNs in the field include: markets, crops and target insects, formulation and shelf life, usage directions, technical support, cost and others (Georgis et al. 2006; Lacey & Georgis 2012). However, the for-mulation is one of the most important factors for the successful use of EPNs as microbial insecticides (Grewal 2002). The EPN formulation is a process of the transformation of living entities into a product that can be applied by practical methods. The EPNs are formulated to facilitate their storage, transport and application (Grewal 2002). The formulations are based on the fundamental principle of energy conservation: restricting their movements (physical restraint) or reducing oxygen consumption to induce an anhydrobiotic partial state (Grewal & Georgis 1999). An optimal formulation should show high and consistent quality, easy handling and transport, high effectiveness and an easy way to be applied in the field.

In the last 15 years, several reviews have pointed out the progress of the EPN formulation and applica-tion technology (Grewal 2002; Grewal & Peters 2005; Shapiro-Ilan et al. 2006, 2012). However, the most detailed review on the EPN formulation was conducted by Grewal (2002). Therefore, we consider necessary to perform a new review of the latest developments in EPN formulations.

In this review, we present a critical analysis of the progress and perspective of the EPN formulations in the last years, emphasising a systemic approach to include disciplines such as invertebrate physiology, materials science, and systems modelling.

What is an EPN formulation?

Generally, the components of the formulations are: an active ingredient, a carrier and additives. Active ingredients in the formulations are EPNs, whereas the carriers used are solids, liquids, gels, and cadavers. The additives are various substances with different functions, such as absorbents, adsorbents, emulsifiers, surfactants, thickeners, humectants, dispersants, antimicrobials, and UV-ray protectors (Grewal 2002). The main purpose of the additives used in the formulations has been to increase the survival and maintain the virulence of the EPNs. Below, we present the EPN formulations currently developed.

Formulations for storage and transport

Aqueous suspension. The most common EPN formulation is an aqueous suspension. It has been used mainly for storage, transportation, and applica-tion (Chen & Glazer 2005). Storage temperatures between 4 and 15°C have produced survival times of 6–12 months for steinernema spp. and 3–6 months for Heterorhabditis spp. (Hazir et al. 2003). How-ever, there are many factors that affect their survival time: sedimentation, high oxygen demand, decreased response of some species at low temperatures, sus-ceptibility to microbial contamination, special storage conditions and appropriate concentration for each species (Grewal & Peters 2005). Also, the refrigera-tion requirements increase costs, hinder the transport (Grewal 2002) and involve the use of application equipment with specific requirements (Toepfer et al. 2010; Brusselman et al. 2012; Lacey & Georgis 2012; Beck et al. 2014; Shapiro-Ilan et al. 2015).

Synthetic sponges. The formulation in polyurethane sponges is accomplished by applying an aqueous suspension of 500–1000 IJs/cm2, which results in an amount of 5–25 million IJs per sponge, which is subsequently placed in a plastic bag for storage. The EPNs formulated in sponges achieve a survival time of 1–3 months at 5–10°C (Grewal 2002) and for their release, the sponges are dipped in a bowl with water. This formulation is not appropriate for mass distribution because it needs refrigeration for storage, the release of the EPNs is time and labour demanding and great amounts of sponge waste are generated. For these reasons, this formulation is only used for storage and transport of small quantities of EPNs for the biological control of pests in home gardens in Europe and North America.

Gels. Yukawa and Pitt (1985) described a system for nematode storage and transport. The EPNs were homogeneously mixed with absorbent materials, such as activated carbon powder, to form a cream, but this formulation has presented the drawbacks of high cost, unpleasant handling and low stability at room temperature (Grewal 1998). Subsequently, Bedding and Butler (1994) and Bedding et al. (2000) developed an aggregate using polyacryla-mide, where the EPNs were partially desiccated, but the survival time at room temperature was low and had difficulties to dissolve (Grewal 2002). On the other hand, Georgis (1990) reached a considerable improvement of this formulation through the use of calcium alginate sheets distributed on plastic

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screens for EPN storage. Chang and Gehert (1991) encapsulated steinernema carpocapsae in a matrix of macrogels, a partially hydrogenated vegetable oil paste containing mono- and diglycerides, which significantly prolonged the viability of the EPNs. The same authors (Chang & Gehert 1992) also developed a paste formulation in which the EPNs were mixed in hydrogenated oil and acrylamide, achieving 80% survival of s. carpocapsae after storage for 35 days at 24–35°C. However, this survival time is considered commercially unacceptable.

Alginate capsules represented an important advance in the EPN formulation. Chen and Glazer (2005) encapsulated steinernema feltiae in this material, which was exposed to osmotic treatment before formulation, with 99.8% survival after 6 months at 23°C and 100% relative humidity. Goud et al. (2010) encapsulated Heterorhabditis indica with different concentration of EPNs and at different temperatures. In the research, the best combination of tempera-ture, population density, and storage was 10°C, up to 1000 IJs per capsule and 90 days of storage. How-ever, better understanding of nematode behaviour, physiology and biochemical process could lead to the further improvement of this formulation. Hussein and Abdel-Aty (2012) encapsulated nematodes of Heterorhabditis bacteriophora and s. carpocapsae in calcium alginate with survival values higher than 50% after 40 days. In general, with alginate capsules survival times up to 6 months at 25°C have been reached (Chen & Glazer 2005). Nevertheless, the extraction steps are time-consuming and expensive because a large number of screens and plastic con-tainers are required, which makes this formulation unsuitable for large-scale applications (Grewal 2002). Also, in agricultural areas where there is not spray equipment availability, the use of this formula-tion presents serious difficulties.

Clay and powder. Bedding (1988) encapsulated s. feltiae, steinernema bibionis, steinernema glaseri, and Heterorhabditis heliothidis in a hygroscopic attapulgite clay formulation with survival time of 8 weeks at 23°C. The formulation was called a “sand-wich” type, because the EPNs are stored between two layers of clay. Products with this formulation were sold, but soon were discontinued due to poor storage stability, clogging of the spray nozzles, and a low nematode-clay proportion (Grewal 2002). Simultaneously, Capinera and Hibbard (1987) developed pellets consisting of a mixture of alfalfa meal, wheat flour, wheat bran, corn oil, and water.

They encapsulated s. feltiae and achieved a mortal-ity of 78.1% on Melanoplus spp. under field condi-tions. Furthermore, Connick et al. (1993) developed wheat flour granules (Pesta) with s. carpocapsae, but achieved a low survival rate after 6 weeks of storage at 21°C. Besides it was observed that wheat flour and high relative humidity promoted the growth of fungi and bacteria. Therefore, Connick et al. (1994) added 0.2% formaldehyde to the mixture of wheat flour, ben-tonite, kaolin, and peat. They stored s. carpocapsae formulated as “improved” Pesta during 26 weeks at 21°C, obtaining 100% mortality of wax moth. These granules were evaluated by Nickle et al. (1994) in a greenhouse against corn rootworm and potato beetle larvae, achieving the 90% control. Silver et al. (1995) encapsulated the EPNs s. carpocapsae, s.  feltiae, steinernema scapterisci, and steinernema riobravis in granules with diatomaceous earth, hydroxyethylcel-lulose, amorphous silica, fumed hydrophobic silica, lignosulfonate, starch, pregelatinised starch, and pregelled attapulgite clay achieving 90% survival after storage for 6 weeks at 25°C.

An increase of the survival time of s. carpocapsae was achieved with the dispersible granules (WG) developed by Grewal (2000a) with a survival rate greater than 80% and infectivity greater than 60% after 5 months storage at 25°C. Grewal (2000b) stored s. carpocapsae, s. feltiae and s. riobravis for 5 months achieving 90% survival at 25°C. Recently, Matadamas-Ortiz et al. (2014) encapsulated s. gla-seri in diatomaceous earth pellets (Celite 209) by means of the downward vertical flow of hygroscopic clays in converging hoppers, with storage conditions at room temperature and ambient moisture, and re-ported 56% survival after 14.1 days. They proposed the mechanisation of the pellet production process. Finally, Cortés-Martínez et al. (2016) evaluated the effect of moisture evaporation from the dia-tomaceous earth pellets on the survival time and infectivity of s. glaseri stored at room temperature and high relative humidity. Also, they evaluated a moisture transfer model by diffusion and evapora-tion to describe the temporal behaviour of moisture content in diatomaceous earth pellets. It was found that the loss of moisture content by evaporation was related to the reduction of storage stability of the IJs in diatomaceous earth pellets.

In review, we verified the microstructure of pellets produced mechanically by Matadamas-Ortiz et al. (2014) with scanning electron microscopy (Figure 1), where we observed that the particle form-size and

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pore characteristics are not uniform. Thus, the sur-vival time of the EPNs in granular reservoirs could be improved by controlling the moisture content of the granular structure. When the pellets have a lower rate of moisture loss, the EPNs can enter to a partial state of anhydrobiosis and therefore increase the survival time. Anhydrobiosis is considered as an important means of achieving the storage stability of entomopathogenic nematodes and is defined as a reversible and physiologically arrested state of dormancy induced by dehydration (Barrett 1991; Grewal 2000b; Grewal et al. 2006). Thus, mois-ture content and moisture loss rate of the pellets are essential research topics to enhance the storage stability of pellet formulations under conditions of room temperature and for retaining the biological control abilities of the EPNs (Grewal & Peters 2005; Perry et al. 2012; Cortés-Martínez et al. 2016).

In the pellet formulation, the factors extremely im-portant for the survival time and the infectivity of the EPNs are: (a) properties of the liquid medium, especially the viscosity and pH of the suspension, (b) granular material properties: size and particle distribution, hy-groscopicity, pH, and density, (c) physical phenomena present in the pellet: moisture and oxygen transfer, (d) storage conditions: temperature and relative humidity, and (e) elaboration method of the pellets (compactation and mechanical stress). All these factors contribute to design an encapsulation process that provides an adequate production rate, opportunity, accessibility, low cost and high quality. Therefore, it is necessary to involve disciplines of systems modelling that can predict the moisture content and moisture loss rate starting from structural properties (size and particle size distribution, hygroscopicity, pH of the material, and

density) and properties of their components (aqueous solution and additives) and based on the knowledge of the moisture requirements by EPNs to achieve a partial state of anhydrobiosis.

Formulations for direct application in the field

Gel. With the aim of eliminating the disadvantages of releasing the EPNs from the alginate granules with sodium citrate, Kaya and Nelsen (1985) encapsu-lated the EPNs s. feltiae and H. heliothidis in calcium alginate granules coated by lipid membranes and fed to larvae of spodoptera exigua Hübner. While feed-ing on the capsules, the larvae released the EPNs. When moisture was present, larval mortality was nearly 100%. To evaluate another possibility of ap-plying directly the alginate granules, Kaya et al. (1987) developed sodium alginate granules with s. feltiae nematodes and a tomato seed in the same conglomerate; as the seed came in contact with moisture, the seed germinated and destroyed the granule structure, releasing the EPNs, and causing 100% mortality of galleria mellonella L. larvae. Also, Navon et al. (1998) encapsulated s. riobravis in edible-to-insects calcium alginate gel and yeast extract used as a phagostimulant to improve the relative consumption rate and digestibility by spo-doptera littoralis Boisduval larvae, obtaining a 100% control. Afterwards, Navon et al. (2002) encapsulated s. carpocapsae in an edible-to-insects gel to control Helicoverpa armigera Hübner and s. littoralis, at a concentration of 1000 s. carpocapsae IJs/g, which caused 95% mortality in H. armigera and 100% in s. littoralis larvaes..

Figure 1. (A) Distribution of the particles and (B) variation of the particle size of the pellets. These were made with 100% diatomaceous earth

(A) (B)

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Due to the interest in direct application of EPNs in the field, Hiltpold et al. (2012) encapsulated EPNs in an alginate shell based on reverse spherification principles. First, a solution with a high concentration of Ca2+ ions is dripped in an alginate bath, resulting in the formation of a spherical shell surrounding a liquid core. The liquid core was obtained by mixing demineralised water with gluconolactate where the nematodes H. bacteriophora were poured. Addition of xanthan gum increased the viscosity of the solution to help in the formation of spheres. After the formation of the spheres, the shell of the capsules was allowed to polymerise. In the field tests, the encapsulated H. bacteriophora nematodes were more effective to control diabrotica virgifera virgifera LeConte than water sprays over the soil surface. However, EPNs were found to escape readily out of the capsules within a few days after encapsulation especially when stored at room temperature, which is inappropriate for commercialization. To overcome this drawback, Kim et al. (2015) modified the capsule properties by changing the reaction temperature for the capsule formation and by adopting the post-treatment of alginate capsules with excessive Ca2+, and then evalu-ated the capacity of the EPNs to escape from the capsules manufactured under different conditions.

This formulation represented a step forward to direct applications in the field. In order to obtain a marketable product, we consider necessary to retain EPNs inside the capsule until needed, maintain EPN infectivity during storage, increase the survival time of the EPNs in the granules, investigate methods for the mass production of granules, evaluate methods to release the EPNs in the soil and the possibility of increasing the number of EPNs per granule.

Infected cadavers. The cadavers are another way to apply EPNs in the field (Shapiro-Ilan et al. 2001, 2008; Shapiro et al. 2003; Bruck et al. 2005; Del Valle et al. 2008a, b; Lacey et al. 2010; Raja et al. 2015). In this formulation, the insect cadaver serves as a reservoir to store the EPNs and then they are applied in the field. Laboratory tests have indicated that this method of application pro-duces a better distribution of the EPNs in the soil than that obtained with the aqueous solution (Sha- piro & Glazer 1996), increases infectivity (Shapiro & Lewis 1999) and is more effective (Shapiro-Ilan et al. 2003). However, it presents a drawback that during storage and transport the larvae can stick or break, which affects the effectiveness of the EPNs (Shapiro-Ilan et al. 2001).

To solve this problem, insect cadavers with coatings were applied to facilitate storage and transportation. Ansari et al. (2009) used a kaolin-starch mixture and Del Valle et al. (2009) used unflavoured gelatin; both authors demonstrated that the coating provided protection and promoted the conservation of the insect cadavers. Another alternative was developed by Shapiro-Ilan et al. (2010) and Morales-Ramos et al. (2013). They designed an automatic packaging machine to wrap EPNs-infected cadavers in masking tape. When it was evaluated with tenebrio molitor L. cadavers, no damage was caused to the EPNs. This mechanical-electronic prototype facilitates the mass packaging of the IJs, reduces work in the process and standardizes the final product (Figure 2). A year later, Spence et al. (2011) developed a technology that in-volved desiccation of g. mellonella larvae to facilitate the transport and handling of EPNs-infected cadavers. Simultaneously, Deol et al. (2011) demonstrated the possibility of directly delivering EPN-infected cadavers through the commercial culture medium Miracle-Gro®. Also, Zhu et al. (2011) developed a mechanised system for the application of infected cadavers in the field (Figure 3). Wang et al. (2014) developed techniques for desiccation and cold storage to promote the mass production and application of EPN-infected cadavers in the field. Most recently, a new application approach was performed to apply EPNs to the field. This ap-proach consists in the releasing of live insect hosts that were pre-infected with the EPN s. carpocapsae against insect pests living in cryptic habitats. The

Figure 2. An automated formulation and packaging machine for enclosing nematode infected hosts in tape (Reprinted with permission from Elsevier)The photograph is a close up with labeled parts including: (A) tape dispenser, (B) cadaver positioning device, (C) cadaver wrapper, (D) moving piston, and (E) cadaver holding bowl (Shapiro-Ilan et al. 2010)

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approach was tested using two model insect pests: a chestnut tree pest, the goat moth Cossus cossus L., and a lawn caterpillar, spodoptera cilium Guenée. C. cossus is considered a pest hard-to-reach via aqueous spray and s. cilium is more openly exposed in the environ-ment. The percentage larval mortality of C. cossus was superior to the application in aqueous spray and the mortality of s. cilium was equal to the aqueous spray method. This approach showed an immense potential to control insect pests living in hard-to-reach cryptic habitats (Gumus et al. 2015).

EPN-infected cadavers have proved to be a good alternative to the direct application of EPNs for the control of soil pests, especially on the basis of the results of Ansari et al. (2009), Del Valle et al. (2009), Shapiro-Ilan et al. (2010), and Spence et

al. (2011) to solve the problem of the fragility of the cadavers. In order to obtain an optimal product, we consider necessary: increasing the survival time at room temperature, standardising the final product and evaluating these technologies with other nematode species of economic importance for crop protection.

The formulations of EPNs have presented a great progress in the pest control in the field. However, the reality is that successful application of EPNs depends on several critical factors such as: ultraviolet radia-tion, adequate soil moisture/relative humidity and temperature (Grewal 2002; Shapiro-Ilan et al. 2012). Improved efficiency and survival of EPNs can be achieved through genetic improvement. Improved strains of EPNs may possess several beneficial traits such as environmental tolerance, virulence, repro-ductive capacity (Strauch et al. 2004; Mukuka et al. 2010; Anbesse et al. 2012; Shapiro-Ilan et al. 2012). Besides, the isolation of native strains resistant to specific conditions of temperature and humidity is an approach that can enhance efficacy in the field (Emelianoff et al. 2008; Malan et al. 2011). In the future, it will be important to develop massive characterisation and selection techniques to obtain the fittest EPNs for formulation.

Conclusions and Remarks

The use of EPNs as biopesticides against insect pests has grown rapidly in recent years. Since the late 1970s, the formulations have focused on increasing the storage time and preserving EPN infectivity in the field. In this review, we identified two types of EPN formulations:

Figure 3. The complete delivery system mounted to one side of the tractor (Zhu et al. 2011) (Reprinted with per-mission of the first author Zhu H.)(1) blower with air controller; (2) metering unit; (3) cadaver hopper; (4) roller chain; (5) nematode infected desiccated cadavers; (6) press-drive wheel; (7) packer wheel; (8) cadaver discharging tube; (9) double disk opener

Table 1. Nematode producers in Europe and in the USA. The table was taken from Kaya et al. (2006). However, in this table we present only the producers that we ratified that are currently operating

Country Company Formulation Nematode species

Germany BASF polymer, clay Ph, Sf, Sc, Hb, Sk,e-nema GmbH clay, polymer Sf, Hb, Sc,

Switzerland Andermatt Biocontrol clay Hb, Sc, SfThe Netherlands Koppert clay, Polymer Sf, Hb, Sc, S sp.

USA

Asa Jung Laboratory clay Sc, SfBioLogic bulk, dispersable granule, sponge, granular Sc, Sf, Hb

Hydro-Gardens sponge Sc, Sf, S sp., H sp.M & R Durango sponge Sc, Sf, Hb

Ph – Phasmarhabditis hermaphrodita; Sf – s. feltiae; Sc – steinernema carpocapsae; Hb – Heterorhabditis bacteriophora; Sk – s. kraussei; S sp. – steinernema species; H sp. – Heterorhabditis species

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(1) Formulations for storage and transport, results show great progress in the EPN survival time, from 7 to 180 days with water dispersible granules and calcium alginate capsules. Commercial products are distributed for storage and transport of nematodes in Europe and North America (Kaya et al. 2006). In Table 1 the EPN producers in Europe and in the USA are listed, but several of them require refrigeration and spraying equipment for application in the field.

(2) Formulations for direct application in the field are the most promising and provide the abil-ity to directly apply EPN-based biopesticides for the control of insect pests. EPN-infected cadavers have positioned as the most viable formulation to apply EPNs for the pest control in the field. Also, Hiltpold-alginate granulation begins to position it-self as a great alternative to apply EPNs in the field. However, it is still possible to improve the granular (pellet with clay and powder) formulation to design an optimal microstructured reservoir for storage, transport, and direct application of EPNs. Also, it is necessary to study the materials taking into ac-count their physical and chemical interaction with the other factors of the formulation by changing the interacting conditions. To identify materials that can gradually induce the nematodes into a partial state of anhydrobiosis. It will be necessary to involve materials science, systems modelling disciplines and mechanical standardised process.

From 2010 to 2015, several studies were developed to facilitate storage and transportation of the EPN-infected cadavers, such as: coating and desiccation of the EPN-infected cadavers and prototype design to wrap EPN-infected cadavers in masking tape. Also, a new formulation was developed (Hiltpold-alginate granulation) as a great alternative to apply EPNs in the field. In general, the future research should be focused on the study of the formulations through a systematic approach to identify new and safe materials that can increase survival time and retain infectivity, and thus obtain an optimal for-mulation (standardisation of the final product) for direct application in the field. Finally, to achieve the best efficiency in the application of EPNs in the field is necessary to have more knowledge of the nematode physiology, soil ecology, systems model-ling disciplines, materials science and other factors related with the farmer, such as the technological and socio-economic context and cultural conditions which may hinder the adoption of EPN technology for the widespread control of agricultural pests.

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Received: 2016–03–07Accepted after correction: 2016–08–11

Published online: 2016–11–08